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Korol, S. V., Jin, Z., Jin, Y., Bhandage, A. K., Tengholm, A., Gandasi, N. R., . . . Birnir, B. (2018). Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells. EBioMedicine, 30
Open this publication in new window or tab >>Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells
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2018 (English)In: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 30Article in journal (Refereed) Published
Abstract [en]

In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting β cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet β cells as biological sensors and reveal that 100-1000nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets β cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D.

Keywords
GABA, GABA(A) receptor, Pancreatic islet, Type 2 diabetes
National Category
Other Medical Sciences not elsewhere specified Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-348267 (URN)10.1016/j.ebiom.2018.03.014 (DOI)000430303000032 ()29606630 (PubMedID)
Funder
Swedish Research Council, 521-2009-4021EXODIAB - Excellence of Diabetes Research in SwedenSwedish Child Diabetes FoundationSwedish Diabetes AssociationNovo NordiskSwedish Society for Medical Research (SSMF)Swedish Research Council, 521-2012-1789Swedish Research Council, 2015-02417Swedish Research Council, 2017-00956Swedish Research Council, 2014-2575
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2018-04-11 Created: 2018-04-11 Last updated: 2018-06-19Bibliographically approved
Gandasi, N. R., Yin, P., Omar-Hmeadi, M., Ottosson Laakso, E., Vikman, P. & Barg, S. (2018). Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes. Cell Metabolism, 27(2), 470-478
Open this publication in new window or tab >>Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
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2018 (English)In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, no 2, p. 470-478Article in journal (Refereed) Published
Abstract [en]

Glucose-stimulated insulin secretion is biphasic, with a rapid first phase and a slowly developing sustained second phase; both are disturbed in type 2 diabetes (T2D). Biphasic secretion results from vastly different release probabilities of individual insulin granules, but the morphological and molecular basis for this is unclear. Here, we show that human insulin secretion and exocytosis critically depend on the availability of membrane-docked granules and that T2D is associated with a strong reduction in granule docking. Glucose accelerated granule docking, and this effect was absent in T2D. Newly docked granules only slowly acquired release competence; this was regulated by major signaling pathways, but not glucose. Gene expression analysis indicated that key proteins involved in granule docking are downregulated in T2D, and overexpression of these proteins increased granule docking. The findings establish granule docking as an important glucose-dependent step in human insulin secretion that is dysregulated in T2D.

Keywords
GLP-1, biphasic secretion, dense core vesicle, docking, exocytosis, genome-wide association, insulin secretion, priming, somatostatin, type 2 diabetes
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-341518 (URN)10.1016/j.cmet.2017.12.017 (DOI)000424465200021 ()29414688 (PubMedID)
Funder
Swedish Research CouncilSwedish Diabetes AssociationSwedish Society for Medical Research (SSMF)The Swedish Brain FoundationNovo NordiskErnfors Foundation
Available from: 2018-02-09 Created: 2018-02-09 Last updated: 2018-03-22Bibliographically approved
Omar-Hmeadi, M., Gandasi, N. R. & Barg, S. (2018). PtdIns(4,5)P2 is not required for secretory granule docking. Traffic: the International Journal of Intracellular Transport, 19(6), 436-445
Open this publication in new window or tab >>PtdIns(4,5)P2 is not required for secretory granule docking
2018 (English)In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 19, no 6, p. 436-445Article in journal (Refereed) Published
Abstract [en]

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P-2) by recruitment of a 5-phosphatase strongly inhibited Ca2+-dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by -toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P-2 accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.

Keywords
exocytosis, insulin, live cell imaging, phosphoinositides, PtdIns(4, 5)P-2, syntaxin clustering, vesicle docking
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356858 (URN)10.1111/tra.12562 (DOI)000432037000005 ()29542271 (PubMedID)
Funder
Swedish Research CouncilSwedish Child Diabetes FoundationSwedish Society for Medical Research (SSMF)Novo NordiskThe Swedish Brain FoundationErnfors Foundation
Available from: 2018-08-15 Created: 2018-08-15 Last updated: 2018-08-15Bibliographically approved
Gandasi, N. R., Yin, P., Riz, M., Chibalina, M. V., Cortese, G., Lund, P.-E., . . . Barg, S. (2017). Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes. Journal of Clinical Investigation, 127(6), 2353-2364
Open this publication in new window or tab >>Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes
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2017 (English)In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 127, no 6, p. 2353-2364Article in journal (Refereed) Published
Abstract [en]

Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II-III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

National Category
Cell and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-321935 (URN)10.1172/JCI88491 (DOI)000402620800029 ()28481223 (PubMedID)
Funder
Swedish Research CouncilSwedish Diabetes AssociationThe Swedish Brain FoundationSwedish Child Diabetes FoundationEXODIAB - Excellence of Diabetes Research in SwedenNovo Nordisk
Available from: 2017-05-12 Created: 2017-05-12 Last updated: 2018-03-11Bibliographically approved
Salunkhe, V. A., Ofori, J. K., Gandasi, N. R., Salo, S. A., Hansson, S., Andersson, M. E., . . . Eliasson, L. (2017). MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles. Physiological Reports, 5(21), Article ID e13493.
Open this publication in new window or tab >>MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles
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2017 (English)In: Physiological Reports, E-ISSN 2051-817X, Vol. 5, no 21, article id e13493Article in journal (Refereed) Published
Abstract [en]

MicroRNAs contribute to the maintenance of optimal cellular functions by fine-tuning protein expression levels. In the pancreatic beta-cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates beta-cell dysfunction, and have earlier shown that islets from the diabetic GK-rat model have increased expression of miRNAs, including miR-335-5p (miR-335). Here, we aim to determine the specific role of miR-335 during development of T2D, and the influence of this miRNA on glucose-stimulated insulin secretion and Ca2+-dependent exocytosis. We found that the expression of miR-335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR-335 in human EndoC-beta H1 and in rat INS-1 832/13 cells (OE335) resulted in decreased glucose-stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single-cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY-mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca2+ current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first-phase insulin secretion during prediabetes. Here, we show a specific role of miR-335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR-335 has the capacity to modulate insulin secretion and Ca2+-dependent exocytosis through effects on granular priming.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
Keywords
Beta cell, exocytosis, insulin secretion, microRNA, patch-clamp, SNAP25, STXBP1, TIRF, Type 2 Diabetes
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-339717 (URN)10.14814/phy2.13493 (DOI)000415351500008 ()
Available from: 2018-01-26 Created: 2018-01-26 Last updated: 2018-01-26Bibliographically approved
Marshall, M., Lund, P.-E. & Barg, S. (2017). Molecular Mechanisms of V-SNARE Function in Secretory Granule Exocytosis. Paper presented at 58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA. Biophysical Journal, 112(3), 395A-395A
Open this publication in new window or tab >>Molecular Mechanisms of V-SNARE Function in Secretory Granule Exocytosis
2017 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 395A-395AArticle in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
CELL PRESS, 2017
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-332760 (URN)000402375600962 ()
Conference
58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA
Available from: 2017-11-06 Created: 2017-11-06 Last updated: 2017-11-06
Omar-Hmeadi, M., Gandasi, N. R. & Barg, S. (2017). Plasma membrane PI(4,5)P-2 is critical for secretory granule exocytosis. Paper presented at ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA.. Molecular Biology of the Cell, 28(26), 3727-3727
Open this publication in new window or tab >>Plasma membrane PI(4,5)P-2 is critical for secretory granule exocytosis
2017 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Philadelphia: American Society for Cell Biology (ASCB), 2017
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-352940 (URN)10.1091/mbc.E17-10-0618 (DOI)000426664300402 ()29237772 (PubMedID)
Conference
ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA.
Note

See: Supplementary Material, 2017 ASCB-EMBO Meeting-Poster Abstracts, p. Sunday-258-Sunday-259, Meeting Abstract: P1403.

Available from: 2018-06-12 Created: 2018-06-12 Last updated: 2018-06-12Bibliographically approved
Alenkvist, I., Gandasi, N. R., Barg, S. & Tengholm, A. (2017). Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis. Diabetes, 66(10), 2610-2622
Open this publication in new window or tab >>Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis
2017 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 66, no 10, p. 2610-2622Article in journal (Refereed) Published
Abstract [en]

Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including -cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the -cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled-Egl-10-pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.

National Category
Clinical Medicine
Identifiers
urn:nbn:se:uu:diva-336299 (URN)10.2337/db17-0050 (DOI)000411195800009 ()28679628 (PubMedID)
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2018-01-23 Created: 2018-01-23 Last updated: 2018-01-23Bibliographically approved
Barg, S. & Gucek, A. (2016). How Kiss-and-Run Can Make Us Sick: SOX4 Puts a Break on the Pore. Diabetes, 65(7), 1791-1793
Open this publication in new window or tab >>How Kiss-and-Run Can Make Us Sick: SOX4 Puts a Break on the Pore
2016 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 65, no 7, p. 1791-1793Article in journal, Editorial material (Other academic) Published
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-300622 (URN)10.2337/dbi16-0019 (DOI)000378463000005 ()27329955 (PubMedID)
Available from: 2016-08-10 Created: 2016-08-10 Last updated: 2017-11-28Bibliographically approved
Alenkvist, I., Gandasi, N. R., Barg, S. & Tengholm, A. (2015). Activation-dependent translocation of Epac2 to granule docking sites at the beta cell plasma membrane. Paper presented at 51st Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 14-18, 2015, Stockholm, SWEDEN. Diabetologia, 58(Suppl. 1), S210-S210
Open this publication in new window or tab >>Activation-dependent translocation of Epac2 to granule docking sites at the beta cell plasma membrane
2015 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 58, no Suppl. 1, p. S210-S210Article in journal, Meeting abstract (Other academic) Published
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-264887 (URN)000359820901111 ()
Conference
51st Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 14-18, 2015, Stockholm, SWEDEN
Note

Meeting Abstract: 421

Available from: 2015-11-05 Created: 2015-10-19 Last updated: 2017-12-01Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-4661-5724

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